Lymphangioleiomyomatosis (LAM) is a genetic disorder characterized by benign lesions of smooth muscle-like LAM cells in the airways and blood and lymph vessels of the lung. The molecular mechanisms triggering LAM cell proliferation remain largely unknown. Genetic studies demonstrated that somatic mutations in the tumor suppressor TSC2 gene are associated with pulmonary LAM. Our preliminary studies demonstrate, for the first time, that without extracellular stimuli loss of tuberin induces the constitutive activation of p70 S6 kinase (p70S6K) and the constitutive phosphorylation of ribosomal protein S6. In this application, we propose to dissect the intracellular molecular signaling mechanisms by which tuberin deficiency, in the absence of extracellular stimuli, leads to abnormal cell cycle progression and proliferation, focusing on defining the roles of the functional domains of tuberin as well as interactions with p70S6k, Rab5, and mTOR. The central hypothesis of the proposal is that loss of the tuberin function promotes LAM cell cycle progression and LAM cell proliferation by the constitutive activation of p70S6K. Rab5 and mTOR serve as downstream effectors of tuberin and upstream modulators of the constitutive activation of p70S6K. To test this hypothesis, in Aim 1, we will determine the role of the structural/functional domains of tuberin in the constitutive activation of p70S6K and cell proliferation. In Aim 2, the role of Rab5 in modulating cell cycle progression and proliferation of tuberin-deficient cells by inducing the constitutive activation of p70S6K, will be examined. In Aim 3, the role of mTOR in modulating the constitutive activation of p7036k will be established. Transfection, microinjection, flow cytometry, immunocytochemistry, immunoblot, DNA synthesis analysis, and Rab5, p70S6K, mTOR activity assays will be utilized to dissect these signaling pathways. These studies will provide insight into the molecular mechanisms that may be important in the pathogenesis of LAM; this understanding may lead to new therapeutic approaches to treat this devastating disease and to improve understanding of the molecular signaling pathways regulating mesenchymal cell proliferation.